Improved Cultivation and Isolation of Diverse Endophytic Bacteria Inhabiting Dendrobium Roots by Using Simply Modified Agar Media

ABSTRACT Dendrobium plants are members of the family Orchidaceae, many of which are endangered orchids with ornamental and medicinal values. Dendrobium endophytic microbes have attracted attention for the development of strategies for plant protection and utilization of medicinal principles. However, the role of endophytic bacteria is poorly elucidated due to the lack of their successful cultivation. This study obtained a total of 749 endophytic isolates from Dendrobium roots using solid media prepared by simply modified methods (separate sterilization of phosphate and agar [PS] and use of gellan gum as a gelling reagent [GG]) and by a conventional method of autoclaving the phosphate and agar together (PT method). Notably, based on a comparison of 16S rRNA gene sequences between the isolates and the Dendrobium root endophyte community, we successfully retrieved more than 50% (17 out of 30) of the predominant endophytic bacterial operational taxonomic units (OTUs) using PS and GG media, which is a much higher recovery rate than that of PT medium (16.7%). We further found that a number of recalcitrant bacteria, including phylogenetically novel isolates and members of even the rarely cultivated phyla Acidobacteriota and Verrucomicrobiota, were obtained only when using PS and/or GG medium. Intriguingly, the majority of these recalcitrant bacteria formed colonies faster on PS or GG medium than on PT medium, which may have contributed to their successful isolation. Taken together, this study succeeded in isolating a wide variety of Dendrobium endophytic bacteria, including predominant ones using PS and GG media, and enables performance of future studies to clarify their unknown roles associated with the growth of Dendrobium plants. IMPORTANCE Dendrobium endophytic bacteria are of great interest since their functions may contribute to the protection of endangered orchids with ornamental and medicinal values. To understand and reveal the “true roles” of the endophytes, obtaining those axenic cultures is necessary even in the metagenomic era. However, no effective methods for isolating a variety of endophytic bacteria have been established. This study first demonstrated that the use of simply modified medium is quite effective and indeed allows the isolation of more than half of the predominant endophytic bacteria inhabiting Dendrobium roots. Besides, even phylogenetically novel and/or recalcitrant endophytic bacteria were successfully obtained by the same strategy. The obtained endophytic bacteria could serve as “living material” for elucidating their unprecedented functions related to the conservation of endangered orchid plants. Furthermore, the culture method used in this study may enable the isolation of various endophytic bacteria dominating not only in orchid plants but also in other useful plants.


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interactions. Recent advances in culture-independent methods, e.g. 16S rRNA gene amplicon 73 sequencing, has made it possible to reveal the composition and diversity of endophytic bacterial 74 community of orchids (2, 7, 15). Metagenomic sequencing approach further allows to predict 75 their functional and metabolic potential. Even though, to understand and verify "true" function 76 and/or discover unprecedented functions of the endophytic bacteria, cultivation and isolation is 77 ultimately essential. To date, however, no effective method obtaining pure culture of a wide 78 variety of bacteria from the interior of plants (not just orchids) has been established. The fact that 79 few environmental bacteria can grow in the laboratory (<1%) has been a limiting factor for 80 unveiling the role of endophytic bacteria (7, 16).

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Several simple methods for preparing media have been devised for widely isolating 82 bacterial groups from the environmental samples, which includes the use of gellan gum as a 83 gelling reagent instead of agar (termed "GG" medium) (17) and separate autoclave sterilization 84 of phosphate and agar (termed "PS" medium, where "S" represents "separately") (18). Both 85 methods are very simple, but those have been found to improve the efficacy of diverse bacterial 86 isolation from several environmental samples including soil, sediment and/or freshwater.

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Furthermore, many reports showed that these techniques are also effective for culturing   Endophytic bacterial communities of the two strains of Dendrobium moniliforme ("GS"; 5 144 isolate of the Verrucomicrobiota was related to the genus Chthoniobacter. In addition, the novel 145 endophytic bacterial isolates (<95% similarity to the validly described strains) were successfully 146 obtained only from PS and GG media (Table 2). All novel isolates were classified into 9 groups 147 based on 98% similarity, which were associated with either Alphaproteobacteria, 148 Gammaproteobacteria, or Bacteroidota. Four or more groups of novel isolates were retrieved 149 from any PS or GG medium. These results indicated that the PS and GG media were more 150 effective for isolating a wide variety of Dendrobium roots-endophytic bacteria including 151 recalcitrant endophytic bacteria such as phylogenetically novel isolates and the rarely cultured 152 phyla Acidobacteriota and Verrucomicrobiota isolates. 153 We further verified how much predominant bacteria of the interior of the Dendrobium 154 roots were retrieved with the media used in this study. For this, the sequence homology between 155 all isolates and the predominant endophytic bacterial OTUs revealed by 16S rRNA gene amplicon 156 sequencing was calculated using BLAST+ (23) and then the isolates exhibiting high homology 157 (>98%) was selected as the isolates affiliated with the predominant OTUs. As a result, a use of 158 PS or GG media allowed to isolate the predominant bacterial groups more widely than the 159 corresponding PT medium (Fig. 2). The recovery rate of the predominant endophytic bacteria by 160 PT-DTS, PS-DTS, GG-DTS, PT-DR2A, PS-DR2A, and GG-DR2A were 13.3%, 46.7%, 23.3%, 161 13.3%, 26.7%, and 23.3%, respectively. Surprisingly, the use of PS and GG media resulted in 162 culturing and isolating more than 50% (17 out of 30) of the predominant OTUs (Fig. 2). Besides, 163 all predominant endophytic isolates obtained using PT media were also retrieved from PS and 164 GG media. The 17 OTUs obtained here consisted of 8 OTUs belonging to Proteobacteria (72.7%), 165 6 OTUs belonging to Actinobacteriota (75%), and 3 OTUs belonging to Bacteroidota (100%), 166 although the predominant OTUs of the phyla Chloroflexi, Verrucomicrobiota, Planctomycetota, 167 Cyanobacteria, Patescibacteria, and Dependentiae were not obtained in this study. Notably, these 168 predominant isolates included the novel endophytic bacterial isolates which were affiliated with 169 OTU404 and OTU1984 (Table 2). Using PS media, we more efficiently isolated members of the 170 predominant bacterial OTUs than using GG media (Fig. 2). However, several predominant 171 isolates (compatible with OTU1537 and OTU1581) were obtained only from GG media. Taken 172 together, we found that high isolation efficacy of the predominant endophytic bacteria could be 173 achieved by using both PS and GG media.

175 Effect of PS and GG methods on growth of recalcitrant bacterial isolates 176
To clarify a reason why the recalcitrant bacterial isolates such as novel isolates (<95% 177 similarity to valid strains) and Acidobacteriota and Verrucomicrobiota isolates were successfully 178 retrieved with PS or GG media but not with PT media (Table 2), we further investigated the effect 179 of PS and GG methods on the colony formation of the endophytic recalcitrant bacterial isolates. 7 216 roots with a relative abundance of more than 1.0% revealed by the MiSeq amplicon sequencing 217 (Fig. 2). To our knowledge, this is an unprecedented level of isolation efficacy of predominant 218 plant endophytic bacterial taxa. These results support that the application of the PS and GG 219 methods is effective for isolating a wide variety of endophytic bacteria including predominant 220 ones from the interior of Dendrobium plants. 221 The previous studies including ours indicated that the application of PS and GG methods 222 is effective for culturing recalcitrant bacteria in soil, sediment, sludge, and/or freshwater (17-21). 223 In accordance with these reports, this study showed that a number of phylogenetically novel 224 isolates (<95% similarity to valid strains) affiliated with phyla Proteobacteria and Bacteroidota, 225 and members of even rarely cultivated phyla Acidobacteriota and Verrucomicrobiota were 226 successfully isolated with PS and/or GG media, whereas these bacteria were not obtained using 227 PT media ( Fig. 1C and Table 2). Notably, the isolates that were affiliated with the novel groups 228 3 and 6 belonged to the predominant endophytic bacterial OTUs (OTU404 and OTU1984) ( Table  229 2). Among nine novel groups, isolates of group 3, 4, 6, and 8 are closely related to plant-derived 230 16S rRNA gene sequence. These suggest that our novel isolates are likely plant-associated (not 231 only with orchids but also with other plant species) . Such success of isolating predominant 232 endophytic bacteria might be brought by growth promotion of the modified cultivation methods 233 adopted in this study. Indeed, the majority of the recalcitrant endophytic bacterial isolates formed 234 visible colonies on PS or GG plates faster than on their respective PT plate (Fig. 3A). In particular, 235 the visible colony formation of the isolate of the phylum Verrucomicrobiota was found at least 236 eight times faster on the PS plate than on the PT plate ( Fig. 3B and C). Similarly, our previous 237 studies reported that the recalcitrant bacterial isolates obtained from soil, sediment, and freshwater 238 grew only or well on PS or GG plates (18-20). For instance, the isolates of rarely cultivated 239 phylum Gemmatimonadota showed a dramatic difference in colony formation on PS/GG plates 240 compared to the corresponding PT plates (18, 20). Collectively, it was suggested that the 241 application of the PS and GG methods might enable cultivation and isolation of recalcitrant 242 bacterial isolates not only from soil, sediment, sludge, and freshwater but also from plant-243 associated (e.g. endophyte) by facilitating their colony formations. 244 To the best of our knowledge, we first succeeded in obtaining bacterial isolates of the 245 phyla Acidobacteriota and Verrucomicrobiota from the internal of orchid plants (Fig. 1C). In 246 particular, Verrucomicrobiota has been rarely isolated from not only the interior of orchids but 247 also that of whole plants. Only two isolates (both belong to subdivision 4, out of seven 248 subdivisions proposed for phylum Verrucomicrobiota) recovered from the root endosphere of 249 Oryza sativa and O. longistaminata were reported (26). The isolate obtained in this study belongs 250 to subdivision 2 (the closest relative is the genus Chthoniobacter) and is phylogenetically 251 different from the abovementioned two isolates (26). This isolate together with the previous ones 8 252 would be useful for elucidating functional roles of Verrucomicrobiota in plant bodies that has 253 long been largely unknown. The phylum Acidobacteriota is composed of diverse members 254 spanning 26 subdivisions and recently has attracted much attention due to its members that are 255 associated with soil-plant ecosystems worldwide (27). For instance, recent studies showed that 256 the non-endophytic isolates affiliated with subdivisions 1, 3, and 6 of phylum Acidobacteriota 257 possessed growth-promoting effects on plants such as Arabidopsis thaliana and duckweed species 258 (28, 29). Since the six isolates obtained in this study belong to subdivision 1 and related to the 259 genera Edaphobacter or Terriglobus, perhaps these isolates might contribute to the growth and 260 development of orchid plants. Accordingly, the endophytic bacterial isolates affiliated with phyla 261 Verrucomicrobiota and Acidobacteriota obtained in the present study must be valuable resources 262 for comprehensive functional analysis of the endophytes of these two phyla. 263 In conclusion, the present study demonstrated that the DTS and DR2A media prepared 264 by PS and GG methods enables the endophytic bacteria isolation from the interior of Dendrobium 265 roots as follows: i) more than 50% of the predominant endophytic bacterial taxa of Dendrobium 266 roots, ii) some phylogenetically novel isolates (<95% similarity to valid strains) affiliated with 267 phyla Proteobacteria and Bacteroidota, and iii) bacteria affiliated with the rarely cultivated phyla 268 Verrucomicrobiota and Acidobacteriota were successfully obtained. These results suggest that 269 the PS and GG methods is effective for widely isolating endophytic bacteria from interior of plant 270 bodies, in addition to soil, sediment and/or freshwater (17, 18, 20). This study used two strains of Dendrobium moniliforme, which had been kindly 279 provided by Mr. Hiroshi Noda and Ms. Hiroko Noda. They characteristically had green stem 280 ("GS" strain) and white stem ("WS" strain), respectively ( Fig. 1A and B), and were cultivated on 281 sphagnum moss under natural light with regular watering for more than two years. Root sections 282 were collected from three plants of each strain and then immediately surface-sterilized as 283 following methods. The roots were treated with 75% ethanol for 30 s; 1 % (v/v) tween 20 for 1 284 min; 3% sodium hypochlorite for 10 min; 75% ethanol for 30 s, and then were rinsed with sterile 285 distilled water (SDW) three times and cut into ca. 0.5 cm long sections with a sterile scalpel. To 286 confirm whether the sterilization process was successful, roots were rolled on the plates of six 287 different media used for the bacterial isolation experiment as described below, and also 100 μL 9 288 of the final water rinse was inoculated and spread on the same plates, which consistently yielded 289 no bacterial colonies incubated at 25 °C for four weeks. For the isolation of endophytic bacteria, 290 one gram of roots was homogenized gently in 9 ml of SDW using a mortar and pestle. To extract 291 DNA from surface-sterilized roots, 0.5 gram of roots was homogenized in liquid nitrogen using 292 a mortar and pestle, and the homogenate was immediately stored at −80°C until use. 293293 294 Endophytic bacterial community analysis by 16S rRNA gene amplicon sequencing 295 A 16S rRNA gene amplicon sequencing was performed to determine predominant 296 endophytic bacterial taxa of both Dendrobium (GS and WS) roots. Genomic DNA was extracted 297 from each homogenized root using FastDNA SPIN Kit for soil (MP Biomedicals, CA, USA) 298 according to the manufacturer's protocol. The DNA extraction was repeated three times. 299 Amplifications of V4 region of 16S rRNA genes were performed using primer set specific for V4 300 region (505F: GTGCCAGCMGCCGCGGTAA; and 806R: GGACTACHVGGGTWTCTAAT). 301 Ex Taq ® DNA polymerase (Takara Bio, Shiga, Japan) was used for PCR amplification and the 302 thermal cycle step was performed with a denaturation step at 94°C for 2 min, followed by 23 303 cycles at 94°C for 30 s, 50°C for 30 s, and 72°C for 30 s and a final extension step at 72°C for 5 304 min. The 16S rRNA gene amplicon libraries were paired-end sequenced on an Illumina MiSeq 305 platform using 2 × 250 bp overlapping paired-end reads (Illumina, CA, USA). Sequence 306 processing was conducted using Qiime2 pipeline (version 2019.7). The paired-end fastq files 307 were demultiplexed with demux-summarize and then were processed by quality filtering, merging 308 of the paired ends and chimera removal with dada2 (30). In dada2 processing, we used options to 309 do the following, 1) primer sequences were removed, 2) forward and reverse reads were truncated 310 to 194 bp and 125 bp, respectively, and 3) the reads containing the base with quality score less 311 than or equal to 15 were truncated. Each read was clustered into operational taxonomic units 312 (OTUs) at 98% similarity using VSEARCH (31). Taxonomy was assigned to each OTU using the 313 SILVA database (silva-138-99) using feature-classifier. Subsequently, reads classified into 314 chloroplasts, mitochondria, and archaea were removed. OTUs with a relative abundance of more 315 than 1.0% either in the interior of GS or WS root were defined as the predominant endophytic 316 bacterial OTUs. Predominant bacterial OTUs were further identified using EzBioCloud server 317 (32), and the corresponding OTUs were excluded when there were no hits. Furthermore, a 318 phylogenetic tree of the predominant endophytic bacterial OTUs was constructed using maximum 319 likelihood (ML) methods. Sequences were aligned using MAFFT with default settings and then 320 a ML tree was constructed using RAxML-NG (version 0.9.0) with 100 bootstrap replicates (33

Effect of PS and GG methods on growth of recalcitrant endophytic bacterial isolates 362
In order to clarify part of reasons why the recalcitrant endophytic bacterial isolates 363 including the phylogenetically novel isolates (<95% similarity to valid strains based on their 364 partial 16S rRNA gene sequence) and the isolates affiliated with members of even rarely 365 cultivated phyla (e.g. Acidobacteriota and Verrucomicrobiota) were obtained from PS or GG 366 media but not from PT media, the effect of PS and GG methods on the colony formation of the 367 recalcitrant isolates were examined based on the criteria of at least twice faster visible colony 368 formation on PS or GG plates than on their respective PT plate, as described by our previous 369 report (20). Each isolate was precultured on the medium plate used for the isolation experiment 370 and then suspended with SDW. Each suspension was spread onto the surface of the medium plate 371 used for the pre-culture and the corresponding PT medium, and then incubated at 25°C under dark 372 conditions. The colony formation was monitored every 18 h by the naked eye and finally by using 373 stereomicroscope.           Thank you very much for serving as the editor for our manuscript (Manuscript #: Spectrum02238-22) and giving us the opportunity to revise it. We also greatly appreciate the careful review and constructive comments from two reviewers. We would like to submit the revised version of manuscript. According to the constructive comments, we have thoroughly revised our manuscript and addressed all the issues raised. We truly believe that we have improved the quality of the manuscript to meet the journal's standards of Microbiology Spectrum. For more information, please confirm the responses to all the comments following this letter.